Purification of human liver cytochrome P-450 and comparison to the enzyme isolated from rat liver

ARCHIVES OF .BIOCHEMISTRY AND BIOPHYSICS

Vol. 199, No. 1, January, pp. 206-219, 1980

Purification of Human Liver Cytochrome P-450 and Comparison to the Enzyme Isolated from Rat Liver1

PHILIP WANG, PATRICIA S. MASON, AND F. PETER GUENGERICH

Department of Biochemistry and Center in Environmental Toxicology,

Vanderbilt University School of Medicine, Nashville, Tennessee 37232

Received July 9, 1979; revised August 22, 1979

Human liver cytochrome P-450 was isolated from autopsy samples using cholate ex-

traction and chromatography on n-octylamino-Sepharose 4B, hydroxylapatite, and DEAE-cellulose gels. Purified preparations contained as much as 14 nmol cytochrome

P-450 rng-’ protein, were free of other hemoproteins, and were active in the mixed- function oxidation of d-benzphetamine and 7-ethoxycoumarin when coupled with either rat

or human liver NADPH-cytochrome P-450 reductase. Some of the preparations were apparently homogeneous as judged by sodium dodecyl sulfate-polyacrylamide gel electro-

phoresis; apparent subunit M,s estimated for several preparations were 53,000 or 55,500. The amino acid composition of one preparation was determined and found to resemble those of rat liver cytochromes P-450, although some variations were noted. Rabbit antibodies

raised to phenobarbital-treated rat liver cytochrome P-450 were more effective in inhibit- ing d-benzphetamine N-demethylase activity in human liver microsomes than were anti-

bodies raised to 3-methylcholanthrene-treated rat liver cytochrome P-450. These antibodies

also inhibited benzo(a)pyrene hydroxylation in human liver microsomes, although the inhibition patterns did not follow a general pattern as in the case of benzphetamine

demethylase activity. Microsomes prepared from three different human liver samples were more effective in eliciting complement fixation with antibodies raised to phenobarbital- than to 3-methylcholanthrene-treated rat liver cytochrome P-450. Complement fixation in

such systems appears to result from similarity of certain rat and human liver cytochrome P-450 antigenic determinants, as fixation could be inhibited by removal of cyto-

chrome P-450-directed antibodies from the total immunoglobulin population and purified human cytochrome P-450 was more effective (on a protein basis) than liver microsomes

in producing fixation. Human liver microsomes prepared from five different individuals all produced 290% complement fixation, but variations were observed in the fixation curves

plotted either Ljers*bs microsomal protein or versus spectrally detectable microsomal cytochrome P-450.

These results indicate that human liver microsomal cytochromes P-450 can be isolated

using modifications of techniques developed for laboratory animals and that human and rat liver cytochromes P-450 share certain features of structural, functional, and immunological similarity. The available data suggest the existence of multiple forms of human liver microsomal cytochrome P-450, but possible artifacts associated with the use of autopsy

samples suggest caution in advancing such a conclusion.

P-4502 plays an important role as the ter- minal oxidase of the microsomal mixed-

I This research was supported by United States

Public Health Service Grants ES 01590 and ES 00267 and Contract NO 1 CP 85672 (National Cancer In- stitute). F.P.G. is the recipient of a United States

Public Health Service Research Career Development Award (ES 00041).

2 Abbreviations used: P-450, liver microsomal cytochrome P-450; SDS, sodium dodecyl sulfate; C’, com-

function oxidase system that functions in the bioactivation and detoxification of a wide variety of potentially toxic xenobiotics

plement; PB, phenobarbital; 3MC, 3-methylcholan-

threne; di-12 GPC, L-cY-dilauroylglyceryl3-phosphoryl- choline; IgG, immunoglobulin G; PB-rat IgG, IgG

raised to P-450 isolated from PB-treated rats; 3MC- IgG, IgG raised to P-450 isolated from 3MC- treated rats.

0003.9861/80/010206-14$02.00/O Copyright 0 1980 by Academic Press, Inc. All rights of reproduction in any form reserved.

206

COMPARISON OF HUMAN AND RAT CYTOCHROMES P-450 207

(l-4). A large body of evidence now indicates that the enzyme exists in multiple forms in several tissues of different experimental animals (1, 5- 10). P-450s have been purified to apparent homogeneity from rabbit liver (1, ll-14), rat liver (5, 7, 10, 11, 15, 16), mouse liver (8), and rabbit lung (17, 18). At this time, much less is known about every aspect of P-450 in humans than in laboratory animals. Most studies with human P-450 have focused upon spectra and activity of microsomal preparations (19-26). Kaschnitz and Coon solubilized human liver microsomes and established the roles of P- 450, NADPH-cytochrome P-450 reductase, and phospholipid fractions in the reconstitution of the mixed-function oxidase system (27). More recently, Kitada and Kama- taki have purified fetal human liver microsomal P-450 to a specific content of as high as 6.7 nmol mg-’ protein and reported some of the spectral properties; these preparations also exhibit activity toward aniline and N-ethylmorphine in the presence of rat liver NADPH-cytochrome P-450 reductase and phosphatidylcholine (28).

Antibodies have been raised to rabbit and rat liver P-450s and used to establish the multiplicity of P-450 (1, 5-7, 16, 29-32), to quantitate individual forms of P-450 (32), to explore the topical arrangement of P-450 in microsomal membranes (30, 33), to establish the role of P-450 in microsomal reactions (34, 35), to compare the P-450s of different tissues and subcellular organelles (35-38), to determine that different P-450s catalyze different reactions involving a single substrate (33, 39), to localize P-450s in tissues (40), and to examine the biosyn- thesis of P-450 (41, 42).” Such immunological studies have been important in aid- ing the understanding of P-450 and utiliza- tion of such techniques could be quite useful in studying various aspects of P-450 in human populations.

This project was undertaken in order to better describe P-450 in humans by purification and using antibodies raised to rat liver enzymes. Methods are presented for the re- producible isolation of human liver P-450

” Ohlsson, R. I., Lane, C., and Guengerich, F. P.. submitted for publication.

in high purity and some chemical, physical, and immunological comparisons to the rat liver enzymes are made.

EXPERIMENTAL PROCEDURES

Preparation of human liver microsomes and en-

zymes. Human liver autopsy samples were obtained 2-8 h after death from the Department of Pathology,

Vanderbilt University. Seven preparations were used in this work: patient 2, 54-year-old male, death due to cardiac arrest; patient 3,70-year-old male, death due to myocardial infarction; patient 4, S-day-old female,

death due to cardiac arrest; patient 5. 69.year-old female, death due to primary breast and ovarian

carcinoma; patient 6, 32.year-old female, death due to gunshot wound; patient 8, Yl-year-old female, death

due to cerebral edema; and patient 9, 14-year-old

male, death due to cardiac arrest. All liver samples were sliced into I-cm sections, washed with a cold

solution of 1.15% (w/v) KC1 containing 0.1 mM

phenylmethylsulfonyl fluoride and 0.1 mM dithiothreitol, blotted free of excess buffer, and stored at -20°C. Microsomes were prepared as described else-

where (36). Specific contents of P-450 for the various microsomal preparations were, respectively (in nmol

P-450 rng-’ protein): patient 2, 0.069; patient 3, 0.13; patient 4. 0.36; patient 5, 0.067; patient 6, 0.18. patient

8, 0.35; and patient 9, 0.17. All purification steps were carried out at 0-4°C‘ and

used potassium phosphate buffers. Microsomes were suspended at a concentration of 2 mg protein ml- ’ in

0.1 M phosphate buffer (pH 7.25) containing 20%’ (v/v) glycerol, 1 mM EDTA, 20 pM butylated hydroxy-

toluene, 0.1 mM phenylmethylsulfonyl fluoride, and 0.1 mM dithiothreitol. A 2Oyc (w/v) solution of re-

crystallized sodium cholate (7) was added to a final concentration of 0.6% (w/v) over 30 min. and the microsomal suspension was centrifuged for 60 min at

105,OOOg. The resulting pellets were pooled and suspended in the original buffer (one-third the original

volume), and cholate was added to a final roncen- tration of 1.54 (w/v) as before. After continued

stirring and centrifugation as before, the pellets were pooled and suspended in the original buffer (one- sixth original volume). Cholate was added to 0.5% (w/v)

and Lubrol PX (Sigma Chemical Co., St. Louis, MO.) was added to 1% (w/v). After stirring for 30 min,

the solution was centrifuged as before; an additional 14% of the P-450 was recovered in the supernatant. The three supernatant fractions were assayed for pro-

tein and P-450; the second fraction (1.5% cholate) was applied to a 2.5 x 40-cm octylamino-Sepharose 4B column which was washed and eluted as described elsewhere (7, 11, 17). The fractions eluted with the

buffer containing 0.33% cholate and 0.06% Emulgen 913 (7, 11. 17) were diluted threefold with a 20% solution of glycerol and applied to a 1 x lo-cm hydroxylapatite (43) column.

208 WANG, MASON, AND GUENGERICH

The hydroxylapatite column was eluted with a step- wise gradient of 20, 40, 80, 150, 300, and 500 mM

potassium phosphate buffers (pH 7.25; 50 ml each buf-

fer); all buffers contained 20% glycerol, 0.1 mM EDTA, and 0.2% (w/v) Emulgen 913. The 300 mM fractions were consistently highest in purity and were used for

all of the experiments described here. Excess deter- gent was removed with Bio-Beads SM-2 (11) unless

otherwise noted; less than 20 /*g of Emulgen 913

remained per nanomole P-450.

In some cases the fractions eluted from hydroxylapatite columns with 150 or 300 mM phosphate

could be further purified by subsequent dialysis against

5 mM potassium phosphate buffer (pH 7.25) containing 20% glycerol, 0.1 mM EDTA, and 0.2% Emulgen 913

and passage through DEAE-Sephacel (5 ml column volume, Pharmacia Fine Chemicals, Uppsala) equili-

brated with the dialysis buffer-the yield for this step was routinely about 50%.

Human liver NADPH-cytochrome P-450 reductase,

assayed as NADPH-cytochrome c reductase, was eluted from the octylamino-Sepharose 4B column after P-450 using a buffer containing 0.35% cholate

and 0.15% deoxycholate (44). The preparation was dialyzed and chromatographed on DEAE-cellulose (45)

and 2’,5’-ADP-agarose (46) gels essentially as de-

scribed elsewhere.

Rat liver enzymes and antibodies. Rat liver P- 450s (“B” fractions) were purified and used to raise

antibodies in female rabbits as previously described (7). IgG fractions were used in all experiments;

preparation was as described elsewhere (7. 30, 36) except that antisera were heated for 20 min at 56°C and then centrifuged for 10 min at 104g prior to

ammonium sulfate fractionation in order to inhibit C’. Specific contents (nmol P-450 mg -’ protein) were 2.0

and 1.4 for PB-treated and 3MCtreated rat livei microsomes, respectively.

Assays. Protein concentrations were estimated

according to Lowry et al. (47). In the case of purified human P-450 preparations. samples were dialyzed versus H,O, concentrated to dryness under

N,, and washed twice with CH,OH to remove deter- gent prior to analysis. Rat P-450 and purified human

P-450s were quantitated according to Omura and Sato (48). The extinction coefficient used was Aerao-r!lo

= 91 mM-l cm-’ (48); subsequent determinations with P-450 purified from patient 6 gave At = 90 mM-’

cm-‘, based upon quantitation of the heme using the pyridine hemochrome assay (Atai7m375 = 32.4 mM-’

cm-’ for reduced versus oxidized pyridine hemochrome) (48, 49). P-450 was assayed in human liver microsomes according to Matsubara et al. (50) be-

cause of hemoglobin contamination. All spectral measurements were made at 20°C using a Cary 219 spectrophotometer in the automatic baseline correction mode. NADPH-cytochrome c reductase activity was assayed (at 30°C) as described elsewhere (45).

Benzphetamine demethylase (51), benzo(a)pyrene hydroxylase (52), 7-ethoxycoumarin 0-deethylase (7), and epoxide (7,8-styrene oxide) hydratase (53) ac-

tivities were assayed as described. Emulgen 913 was estimated as described by Garewal (54).

C’$xatcntion techniques. The basic procedure was used

as described by Levine and his associates (55, 56). Guinea pig C’ was purchased from Cappel Labora-

tories, Cochranville, Pennsylvania and sheep erythrocytes were obtained fresh weekly from Grand Island

Biological Company Diagnostics, Madison, Wisconsin. IgGs and test antigens were diluted in 10 mM

Tris-HCl buffer (pH 7.4) containing 0.14 M NaCl,

0.5 mM MgCl,, 0.15 mM CaCl,, and 1 mg ml-’ bovine serum albumin, and the appropriate volumes of

each were mixed on ice. C’ (0.2 ml of a ca. 11800

dilution) was added along with sufficient diluent to bring the total volume in each tube to 0.8 ml. After 16 h at 4”C, 0.2 ml of diluent was added (to

each tube) containing enough washed erythrocytes to give a final A,,, reading of ca. 1.0. Tubes were incubated for 30 min at 37°C with gentle shaking,

chilled on ice, and centrifuged at IOOOg for 5 min. The supernatants were transferred to clean tubes

and absorbances were measured at 413 nm. Percent-

age C’ fixation (anticomplementarity) was calculated using minus antigen blanks for complete lysis and minus C’ blanks for complete inhibition of lysis:

minus antigen A,,:, ~ observed A,,,

minus antigen A,,, - minus C’ A,,:,

= percentage C’ fixation.

Individual curves presented in each figure were from experiments carried out on single days with single sets of reagents.

RESULTS

Stability of Huma,n Liver Microsomal Enzymes

The amounts of liver needed for the purification of substantial amounts of human enzymes required that autopsy material be used, as samples from different patients were not pooled. Preliminary studies were carried out with portions of the liver of a single patient (No. 9) to determine the stability of the enzymes under consideration (Fig. 1). Epoxide hydratase activity was rather stable, even when liver was kept at 23°C (Fig. 1A); 7,8- styrene oxide hydratase activity had a t1,2 of about 43 h. NADPH-cytochrome c reductase and P-450 were less stable, with respective t ,,2 values of 14 and 8 h, respec-


FIG. 1. Stability of human liver microsomal enzymes under varying conditions. An autopsy sample from patient 9, obtained 3.5 h after death, was divided into several portions (lobes were distributed

randomly) and microsomes were prepared. Assays were carried out as described under Experimental Procedures, except in the case of benzphetamine demethylase activity in part A, where a calorimetric

assay was used as previously described (11) with minus benzphetamine blank corrections; results are expressed relative to activities determined immediately after preparation of microsomes from

freshly procured liver-O.17 nmol P-450 rng-’ protein, 150 t 5 nmol cytochrome c reduced mini rng-’ protein, 19.7 -+ 0.3 nmol 7,8-styrene oxide hydrated min’ mg-’ protein, 0.36 2 0.05 nmol

%hydroxybenzo(a)pyrene formed min’ rng-’ protein, and 0.29 t 0.03 nmol HCHO formed (from benzphetamine) min’ rng-’ protein. When SD is indicated by bars, triplicate experiments were carried out. (A) Portions of intact liver were maintained at 23°C; microsomes were prepared

at the indicated time points and assays were carried out immediately. (B) Portions of intact liver

were maintained at -20°C; microsomes were prepared at the indicated time points and assays were carried out immediately. (C) Microsomes were prepared (at 3.5 h after death of patient), frozen at -2o”C, and assayed at the indicated time points.

tively. Benzo( a)pyrene hydrodroxylase activity was very unstable, having a t1,2 of 1.5 h for the rapid phase of inactivation. However, d-benzphetamine N-demethylase activity was more stable under these conditions (t,,* ea. 30 h).

1 InM EDTA and 20% glycerol) as a function of time after preparation (Fig. 1C). Benzo(a)pyrene hydroxylase activity was actually less stable than in intact liver.

Parallel results were obtained when liver was stored at -20°C (Fig. la), although losses of activity were significantly de- creased and were apparently abolished in the cases of epoxide hydratase and benzphetamine demethylase. The stability of the various enzymes was also examined in microsomes (maintained at -20°C in 10 mM

Tris-acetate buffer (pH 7.4) containing

Purification of Human Liver P-450

The procedures chosen were based upon previous success in the purification of P-450s from rabbit liver (12), rat liver (7, II), and rabbit lung (17). The first attempt resulted in extensive purification of P-450 from patient 3; the nominal specific content was 9.4 nmol P-450 mg-’ protein and the overall yield was 0.7%.


TABLE I

PURIFICATION OF HUMAN LIVER MICROSOMALP-450"

Fraction Protein

(mg) P-450 (nmol)

Apparent Specific

yield content

(%) (nmol mg-‘) Fold

purification

Microsomes 4200 767 (100) 0.18 (1) 0.6% Cholate extract 3200 278 36 0.017 0.5

1.5% Cholate extract 785 217 28 0.28 1.5

Octylamino-Sepharose 4B 41 121 16 2.97 16.2 Hydroxylapatite 2.6 35 4.5 13.1 71.6

a The procedure was carried out using a portion of the liver sample obtained from patient 6. Only the 1.5% cholate extract was used for subsequent steps. Data are shown only for the 300 mM fraction recovered from the

hydroxylapatite column.

Methods were refined as noted under Ex- perimental Procedures to purify P-450 from patient 6 to apparent homogeneity in 4.5% yield (Table I). Three separate purifications with portions of the same liver (patient 6) yielded P-450 ranging in nominal specific content from 9.5-13.8 nmol P-450 mg-’ protein in 1.6-4.5% overall apparent yield; P-450 was also purified from patient 8 to a specific content of 14.1 nmol P-450 mg-’ (0.5% yield).

The P-450s obtained from patients 6 and 8 migrated as single bands upon SDS- polyacrylamide gel electrophoresis as described by Laemmli (57) or as subsequently modified (7). The apparent M,.s were 53,000 d for those preparations (Fig. 2); the major

band in the preparation derived from patient 3 had an apparent M, of 55,500. For comparison, the following apparent M,s were estimated for rat and rabbit liver P-450s (7, 11) in these electrophoretic experiments: PB-treated rat P-450, 53,000; 3MC-treated rat P-450, 55,000; PB-treated rabbit LM-2, 51,500; and P-naphthoflavone- treated rabbit LM-4, 53,500.

Spectra indicated the absence of other hemoproteins (Fig. 3). The oxidized form of the isolated P-450 existed as a low-spin hemoprotein with absorption maxima at 416, 528, and 570 nm. The A39,,LA4,6 ratio was 0.55, as also found with several isolated rat and rabbit liver P-450 preparations (11). Other maxima were observed

I 2 3 4567 8 9 IO

FIG. 2. SDS-polyacrylamide gel electrophoresis of human liver P-450 preparations. Samples were electrophoresed according to Laemmli (57) except that the concentrations of the components of the

cathode buffer were doubled (7). The anode was at the bottom of each of the three gels; gels were stained and destained according to Fairbanks et al. (58). Samples were as follows: 1, 7, and 9, 3.75 pg each of standard bovine serum albumin (accepted M, 68,000), bovine liver catalase (M,

58,000), Escherichia coli L-glutamate dehydrogenase (M, 53,000), hen egg ovalbumin (M, 43,000), and rabbit muscle aldolase (M, 40,000); 2, octylamino-Sepharose 4B fraction from patient 3, 10 pg;

3, hydroxylapatite-DEAE-cellulose fraction from patient 3, 3 pg; 4 -6, hydroxylapatite fraction from patient 6, 5, 10, and 13 Fg, respectively; 8, hydroxylapatite fraction from patient 8, 4.5 pg; and 10, hydroxylapatite fraction from patient 6, 5 Fg.

COMPARISON OF HUMAN AND RAT CYTOCHROMES P-450

FIG. 3. (A) Difference spectrum of purified human liver P-450. A 0.25 pM solution of a final human liver P-450 preparation (patient 6) in 5 mM potassium phosphate buffer (pH 7.7) containing 20%

glycerol. 0.1 mM EDTA, and 0.2% Emulgen 913 was divided into two 0.2ml cuvettes which were equilibrated with CO; a baseline (- - -) was established and Na,S,O? was added to the sample

cuvette (---). (R) Absolute spectra of purified human liver P-450. The sample cuvette contained 0.25 @M purified P-450 (patient 6) in the same buffer as used in part A. The oxidized (---1.

Na,S,O,-reduced (- - -), and reduced-CO (-.-) spectra are shown.

at 418 and 560 nm in the reduced state cated that the estimates of protein con- and 450 (450.0 -+ 0.2) and 552 nm for the centration using the method of Lowry reduced-CO complex. Of all the microsomal et al. (47) were reasonably accurate (i.e., and purified preparations examined, none within 5%). had reduced-CO maxima that were out of the range of 449-451 nm. PuriIcation of Human Liver

The amino acid composition of a human liver P-450 preparation showed some similarity to the major P-450s isolated from PB- and SMC-treated rats (Table II). The most striking difference was in the composition of basic residues; i.e., the human preparation contained very little histidine and much more lysine than either of the two rat P-450s. Difference indices were calculated according to Metzger et al. (60): the two rat liver P-450s had an index of 3.1; the human liver P-450 had an index of 6.3 when compared to the PB-treated rat P-450 and 5.7 when compared to the 3MC- treated rat P-450. These values indicate that the two rat P-450s are more closely related to each other than to the human enzyme. The amino acid analysis data indi-

NADPH-cytochrome P-450 Reductase

The reductase was purified from patient 6 to a specific activity of 53 pm01 cytochrome c reduced min-’ mg-’ protein using techniques developed for the purification of the enzyme from other species (44-46). The enzyme was not homogeneous as judged by SDS-polyacrylamide gel electrophoresis (58), but >80% of the Coomassie blue stain was associated with a single band of apparent M, 70,000 (not shown). This M, was slightly less than that observed for the rat liver enzyme under identical conditions (M, 74,000) (46). The preparation was active in supporting P-450-associated hydroxylation activities (vide infra).


TABLE II Reconstitution of Human Liver

COMPARISON OF AMINO ACID COMPOSITION OF HUMAN ANDRAT LIVER

CYTOCHROMES P-450"

Microsomal Mixed-Function Oxidase Activity

Number of residues per subunit P-450

Amino acid

Pheno- S-Methyl- barbital- cholanthrene-

treated treated Human rat rat

Lysine 46 27 31

Histidine 1 13 13 Arginine 19 25 25

Asx 47 42 43 Threonine 25 29 29

Serine 36 30 38 Glx 45 52 43

Proline 28 27 37 Glycine 45 33 32 Alanine 27 29 26

Valine 22 27 28

Methionine 15 11 10 Isoleucine 28 27 24

Leucine 53 59 54 Tyrosine Phenylalanine 2:

14 14 32 28

Half-cystine 5b 7 6 Tryptophan 4 2 6 Total 474 486 487

The P-450 preparation derived from patient 6 was active toward d-benzphetamine and ‘i-ethoxycoumarin when coupled with either rat or human liver NADPH-cytochrome P-450 recluctase (Table III). The dealkylation rates were higher than those observed with the microsomal preparation when expressed on the basis of P-450. However, benzo(a)pyrene hydroxylase activity was not detected with this same P-450. However, benzo(a)pyrene hydroxylase activity was not detected with this same P-450 preparation. Other experiments incli- catecl that the rates of benzphetamine and 7-ethoxycoumarin metabolism were lowered to less than 11 and 40%, respectively, when di-12 GPC was omitted from the respective systems, and that the addi- tion of Emulgen 913 to 10 pg ml-’ did not affect the rates of metabolism.

Effect of Antibodies upon Activity of Human Liver P-450

0 Data for the rat liver proteins were reported elsewhere (7). The preparation derived from patient 6

(Table I) was hydrolyzed and analyses were done in triplicate essentially as described elsewhere (7).

b Estimated by titration with 4,4’-pyridine di- sulfide (59).

PB rat-IgG was found to inhibit benzphetamine clemethylase activity in microsomes prepared from three different individuals (Fig. 4); the extent of maximum inhibition ranged from 25-65% and was statistically significant when compared to the effect of preimmune IgG. The effect of 3MC rat-IgG was rather small if significant at all.

TABLE III

METABOLISMOF SUBSTRATES BYHUMAN LIVER MICROSOMESAND PURIFIED P-450"

Turnover number

Purified human P-450

Substrate

d-Benzphetamine ‘I-Ethoxycoumarin

Microsomes (min-‘)

2.7 ” 0.20 0.0137 5 0.0006

Plus rat reductase (min-‘)

27.0 k 10.9 0.023 2 0.004

Plus human reductase (min-I)

21.9 t 8.0 0.021 + 0.011

a Assays measuring benzphetamine (51) and 7-ethoxycoumarin (7) activities were carried out in triplicate as described. Incubations contained 0.5 mg microsomal protein or reconstituted systems composed of 5 pmol purified human P-450 (patient 6) plus an equimolar amount of rat (7,46) or human liver NADPH-cytochrome

P-450 reductase and 50 PM di-12 GPC. Turnover numbers (means 2 SD) are expressed as nmol product formed min-’ nmol-’ P-450.


i

0 .i 0

.----L--L --e---F ,-1 2 3 20

6 mg IgG nmol -’ P-450

I 0 --~

0

I ~~~- L-- -‘,----3,.-- --yf-L- i

1 * 5 20

C mg IgG nmol-’ P-450

FIG. 4. Effect of antibodies raised to rat liver P-450s on d-benzphetamine N-demethylase activity of human liver microsomes. Incubations were carried out essentially as described elsewhere (36) using an amount of microsomal protein equivalent to 100 pmol of P-450 in each assay. Either preimmune IgG, IgG raised to PB-treated rat P-450, or IgG raised to 3MC-treated rat P-450 was present at

each indicated concentration. All assays were carried out in triplicate and results are expressed as means 2 SD. The basal levels of HCHO formation were 0.156 C 0.016, 0.60 k 0.06, and 1.67 + 0.12 nmol min-’ nmol-’ P-450 for patients 4 (A), 8 (B), and 9 (Cl, respectively.


The inhibition of benzo(a)pyrene hydroxylation was also examined (Fig. 5). 3MC-rat IgG produced varying levels of inhibition in three different patients. PB rat IgG had no effect in one case (Fig. 4B), inhibited metabolism by 70% in another case (Fig. 4C), and gave some stimulation in the third ease (Fig. 4A).

C’ Fixation Experiments

Preliminary experiments indicated that none of the human liver microsomal preparations yielded immunodiffusion precipitin lines with either PB rat-IgG or 3MC rat IgG when examined under conditions used for rat P-450s (6, ‘7, 36). Complexation of human liver P-450 with PB rat-IgG was

i

detected using C’ fixation techniques, as shown in Fig. 6. Considerably less fixation, if any, was detected using 3MC rat- IgG (Fig. 7).

The C’ fixation activity of the PB rat-IgG was nearly abolished by precipitation of the rat P-450-directed antibodies from the IgG with PB-treated rat liver microsomes as shown in Fig. 8, suggesting that similar immunological determinants are present in rat and human liver microsomal P-450s. The P-450-directed antibodies in the PB rat-IgG preparation could also be partially removed by centrifugation of mixtures of PB rat-IgG and highly purified rat P-450; after such treatment, PB-rat IgG was less effective than untreated PB-rat IgG in C’ fixation using either PB-treated rat microsomes or

FIG. 5. Effect of antibodies raised to rat liver P-450s on benzo(a)pyrene hydroxylase activity of

human liver microsomes. Incubations were carried out essentially as described elsewhere (36) using an amount of microsomal protein equivalent to 100 pmol of P-450 in each assay. All assays were carried

out in triplicate and are expressed as means 2 SD. Either preimmune IgG, IgG raised to PB-treated rat P-450, or IgG raised to 3MC-treated rat P-450 was present at each indicated concentration. The basal levels of 3-hydroxybenzo(a)pyrene formation were 0.201 2 0.085, 1.79 _f 0.23, and 2.65

2 0.12 nmol min-’ nmol-’ P-450 for patients 4 (A), 8 (B), and 9 (C), respectively.


FIG. 6. C’ fixation by PB rat-IgG and PB-treated rat liver or human liver microsomes. Varying levels of PB rat-IgG were mixed with either 0.2 kg of PB-treated rat liver microsomes (A) or 2 pg of liver microsomes derived from each of the following human samples: patient 2 CO), patient 3 (W), or

patient 6 (A). C’ fixation assays were carried out as described under Experimental Procedures.

human microsomes (Fig. 8). The lack of complete inhibition of C’ fixation was due to incomplete precipitation of P-450lIgG complexes at these concentrations, as 30% apparent C’ fixation was found for incubations devoid of microsomes. No greater inhibition of C’ fixation was found with a number of higher or lower P-450:IgG ratios for removal of P-450-directed antibodies. Treatment of the PB rat-IgGIP-450 supernatant with varying levels of a second antibody (swine IgG raised to rabbit IgG) did not specifically precipitate PB rat-IgG/ P-450 complexes.

Highly purified human P-450 was more effective in C’ fixation on a protein basis

than were human microsomes (Fig. 9>, further implicating P-450 in the C’ fixation process. The difference in the curves is not in direct proportion to the purification factor; however, other forms of P-450 may be present in the microsomes that are more similar immunologically.

Differences in C’ Fixation by P-450s of Different Humans

Varying amounts of the different human microsomal preparations were incubated with a fixed amount of PB-rat IgG in C’ fixation studies, as curves from such experiments are more sensitive in detecting

FIG. 7. C’ fixation by 3MC rat-IgG and 3MC-treated rat liver or human liver microsomes. Varying levels of 3MC rat-IgG were mixed with either 0.2 pg of 3MC-treated rat liver microsomes (A) or

2 kg of liver microsomes derived from each of the following human samples: patient 2 (O), patient 3 (m), or patient 6 (A). C’ fixation assays were carried out as described under Experimental Procedures.


FIG. 8. Inhibition of PB-treated rat and human microsomal C’ fixation by removal of P-450. directed antibodies. C’ fixation was carried out as described under Fig. 6 with PB-treated rat liver microsomes (A - A) and human liver microsomes derived from patient 6 (A - A). PB-rat IgG was mixed with either 2 nmol purified PB-treated rat liver P-450 or 1 mg PB-treated rat liver microsomes/mg PB rat-IgG, allowed to stand 1 h at 23°C and 24 h at 4”C, and centrifuged at ZO,OOOg for 30 min. Aliquots of the supernatants were used in parallel experiments without correction for the amounts of IgG removed: 0.2 pg PB-treated rat microsomes plus PB-rat IgG treated with purified PB rat P-450 (A - - - A); 2 *g human microsomes (patient 6) plus PB-rat IgG treated with purified PB rat P-450 (A - - - a), 0.2 gg PB-treated rat microsomes plus PB-rat IgG treated with PB-treated rat microsomes (A -.- A); and 2 pg human microsomes (patient 6) plus PB-rat IgG treated with PB-treated rat microsomes (a -‘- A).

differences in proteins than the type used in Figs. 6 and 7 (55, 56). When the results were plotted as a function of protein used, the order of C’ fixation (most efficient to least) was patient 2 > patient 4 > patient 6 = patient 3 s patient 5 (Fig. 10A). The amount of protein required for 50% C’ fixation differed 12-fold between patients 2 and 5. Plotting the results as a function of spectrally detectable P-450 also showed a 1Zfold difference in the amount

I 1

FIG. 9. Comparison of C’ fixation by human liver microsomes and highly purified human liver microsomal P-450. C’ fixation assays were carried out using 5 pg of PB rat-IgG and varying levels of microsomes derived from patient 6 (A) or P-450 (hydroxylapatite preparation, Table I in duplicate) derived from patient 6 (0).

of P-450 required for 50% C’ fixation (Fig. 10B); the order of C’ fixation was patient 2 + patient 3 > patient 6 = patient 4 > patient 5. In all cases, 290% C’ fixation was achieved at some concentration of microsomes.

DISCUSSION

With some modification, techniques developed for the purification of P-450 and NADPH-cytochrome P-450 reductase from experimental animals have been used to isolate the enzymes from human liver in high states of purity. Although some of the final preparations were apparently homogeneous as judged by SDS-polyacrylamide gel electrophoresis, specific contents of P-450 mg-’ protein were somewhat less than expected on the basis of subunit M, (i.e., 17-19 nmol). The protein estima- tion was not in error, as judged by quan- titative amino acid analysis. Hydrodynamic studies indicate that M, estimates for rat liver P-450s obtained with SDS-polyacrylamide gel electrophoresis are correct (7),4 and we assume that this is the case

d Guengerich, F. P., and Holladay, I>. A., submitted for publication.

COMPARISONOFHUMANANDRATCYTOCHROMESP-450 217

for human liver P-450 as well. The differ-

B HUMeN CYTOCHROME P-450, pmoles

FIG. 10. C’ fixation as a function of human microsomal protein or P-450. C’ fixation assays were

carried out as described under Experimental Procedures with 5 pg of PB rat-IgG and varying amounts of human liver microsomes derived from patient 2 (O), patient 3 (m), patient 4 (O),

patient 5 (O), and patient 6 (a). (A) Data are plotted versus concentration of human liver microsomal protein. (B) Data are plotted versus concentration of human liver microsomal protein. (B) Data

are plotted versus concentration of spectrally detectable human liver P-450.

ence was not due to the presence of cytochrome P-420 (i.e., a form(s) of denatured P-450) (Fig. 3). The discrepancy was also probably not due to error in the extinction coefficient; ~~~~~~~~~ = 91 MM~’ cm-’ has been confirmed with a variety of rat and rabbit preparations (5, 48) and a value of 90 mM-' cm-’ was obtained for a human preparation. Thus, the discrepancy must arise from the presence of apoenzyme or some other protein not resolved by SDS- polyacrylamide gel electrophoresis.

The stability profiles deserve some com- ment. Benzo(a)pyrene hydroxylase activity is not very stable in human liver, in con- currence with a recent report by Prough et al. (26). This is somewhat surprising since Robinson et al. reported that this activity was stable in mice for 7 h after

death (61). Benzphetamine demethylase activity was much more stable under all conditions examined, as is the case for another microsomal enzyme, epoxide hydratase. The data suggest caution in using the measurement of benzo(a)pyrene hydroxylase activity in autopsy samples as an index of metabolism. The results also suggest considerable variation in stabilities of different human liver P-450s and that minor amounts of certain individual forms or sub- populations of P-450 may be responsible for the bulk of either benzo(a)pyrene hydroxylase or benzphetamine demethylase activity.

Although repeated attempts to establish immunochemical similarity of various rat and human P-450s using double-diffusion analysis were negative, similarity was detected using C’ fixation and inhibition of


activity. The general conclusion can be 2. CONNEY, A. H. (1967) Pharmacol. Rev. 19,

drawn that the P-450s in the human 317-366.

liver samples examined here were immuno- 3. GELBOIN, H. V. (1967) Advan. Cancer Res. 10,

logically more similar to P-450 isolated 1-81.

from PB-treated than SMC-treated rats. 4. GILLETTE, J. R. (1966) Advan. Pharmacol. 4,

The apparent trend among various patients 219-261.

was that benzphetamine metabolism was 5. GUENCERJCH, F. P. (19’79) Pharmacol. Ther. A

more readily inhibited by PB-rat IgG and 6, 99-121.

6. THOMAS, P. E., benzo(a)pyrene metabolism was more read-

Lu, A. Y. H., RYAN, D., WEST, S. B., KAWALEK, J., AND LEVIN, W.

ily inhibited by 3MC-rat IgG, which might (1976) Mol. Pharmacol. 12, 746-758.

be expected on the basis of some of the 7. GUENGERICH, F. P. (1978) J. Bi01. Chem. 253,

results obtained with rat liver microsomes 7931-7939.

(31, 33, 36). Other experiments indicate 8. HUANG, M-T., WEST, S. B., AND Lu, A. Y. H.

that benzo(a)pyrene hydroxylase activity (1976) J. Biol. Chem. 251, 4659-4665.

in human lymphocytes and monocytes is 9. ULLRICH, V., AND KREMERS, P. (1977) Arch.

more readily inhibited by 3MC-rat IgG Toxicol. 39, 41-50.

than by PB-rat IgG.” 10. LEVIN, W. (1977) in Microsomes and Drug

In conclusion, the available data suggest Oxidations (Ullrich, V., Roots, I., Hildebrandt, A., Estabrook, R. W., and Conney, A. H.,

that rat and human P-450s are somewhat eds.), pp. 735-747, Pergamon Press, New similar as judged by certain physical and York.

immunological techniques, although differ- 11. GUENGERICH, F. P. (1977) J. Biol. Chem. 252,

ences definitely exist. The results also sug- 3970-3979.

gest that humans contain multiple forms of 12. IMAI, Y., AND SATO, R. (1974) Biochem. Bio-

P-450, as judged by isolation of P-450s phys. Res. Commun. 60, 8-14.

with different apparent M,s (Fig. 2), C’ 13. JOHNSON, E. F., AND MULLER-EBERHARD, U.

fixation studies (Fig. lo), individual differ- (1977) Biochem. Biophys. Res. Commun. 76,

ences in susceptibility to antibodies (Fig. 652-659.

4), differences in the stabilities of P-450- 14. PHILPOT, R. M., AND ARINC, E. (1976) Mol.

catalyzed activities (Fig. l), and the separa- Pharmacol. 12, 483-493.

tion of P-450 into several fractions in the 15. RYAN, D., Lu, A. Y. H., KAWALEK, J., WEST,

various chromatographic procedures. How- S. B., AND LEVIN, W. (1975) Biochem. Bio-

ever, such a conclusion must be considered phys. Res. Commun. 64, 1134-1141.

tentative because of possible artifacts that 16. RYAN, D. E., THOMAS, P. E., AND LEVIN, W.

(1977) Mol. Pharmacol. 13, 521-532.

may be associated with the use of autopsy 17. GUENGERICH, F. P. (1977) Mol. Pharmacol. 13,

material. 911-923.

18. WOLF, C. R., SZUTOWSKI, M. M., BALL, L. M.,

ACKNOWLEDGMENTS AND PHILPOT, R. M. (1978) Chem.-Biol.

We thank Drs. Frank Chytil, Robert Briggs, and Interac. 21, 29-43.

Lubomir Hnilica for their advice in carrying out the 19. KUNTZMAN, R., MARK, L. C., BRAND, L.,

complement fixation work, Dr. John Edland for his JACOBSON, M., LEVIN, W., AND CONNEY, A. H.

help in procuring autopsy samples, and Mr. W. Morgan (1966) J. Phamacol. Ezp. Ther. 152, 151-

Crawford, Jr. and Mrs. Margaret B. Mitchell for 156.

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Documents

Purification of human liver cytochrome P-450 and comparison to the enzyme isolated from rat liver